Target recognition element and biosensor including the same

ABSTRACT

The present invention provides a target recognizing element wherein a receptor is fixed to an inclusion complex containing a mediator. The target recognizing element includes a first host molecule having hydrophilic groups and an inclusion site, a second host molecule having hydrophilic groups and an inclusion site, a receptor which is bonded to the hydrophilic groups of the second host molecule and which reacts with the target, and a guest molecule which is included by the inclusion site of the first host molecule and the inclusion site of the second host molecule and which transfers an electric charge generated by the reaction between the target and the receptor.

TECHNICAL FIELD

The present invention relates to a target recognizing element whichspecifically bonds to a substance to be measured and to a biosensorwhich uses this target recognizing element.

BACKGROUND ART

Biosensors include enzyme sensors, immunosensors, and bacteriologicalsensors, and are considered to be very important for measuring materialproperties or substances (hereinafter referred to as targets) in a widevariety of fields such as the fields of medicine, food, and industry.

For instance, a glucose sensor measures the blood sugar level from theconcentration of glucose in the blood. The glucose sensor has anelectrode and an enzyme membrane which covers the electrode. Glucoseoxidase (GOD) is fixed on the enzyme membrane as a receptor whichspecifically reacts with glucose.

Glucose is oxidized by GOD in the glucose sensor according to thereaction formula (1), and is broken down into gluconic acid and hydrogenperoxide.

(Formula 1)C₆H₁₂O₆+O₂→C₆H₁₀O₆+H₂O₂  (1)

Next, the hydrogen peroxide produced in the reaction formula (1) isdiffused in the solution between the electrode and the enzyme membraneto the electrode surface, and undergoes electrolysis at the electrode asshown in reaction formula (2), and the electrons are transferred to theelectrode.

(Formula 2)H₂O₂→*2H⁺+O₂+2e⁻  (2)

A proportional relationship exists between the glucose concentration,the diffusion of glucose to the enzyme membrane, and the diffusion ofhydrogen peroxide in the solution, and the glucose concentration can beobtained by measuring the electrical current from the electrolysisreaction of reaction formula (2). However, with this type of glucosesensor, the transfer of electrons to the electrode from the enzymemembrane is dependent on the diffusion speed and the diffusionconcentration or the like of the hydrogen peroxide to the electrode, sorapid measurements and measurements with high electrical currents aredifficult.

Therefore, biosensors are being developed which transfer a chargebetween a receptor and the electrode using a mediator. FIG. 7 shows theoperating principle of a mediator type biosensor, and the followingreaction takes place between the mediator M, an enzyme E, and asubstrate S which reacts with the enzyme E.

Electrons are transferred by the oxidation reduction reaction betweenthe oxidized enzyme E_(ox) and the substrate S, and reduced enzymeE_(red) and product P are produced. Next, oxidized enzyme E_(ox) andreduced mediator M_(red) are produced by the oxidation reductionreaction between the reduced enzyme E_(red) and the oxidized mediatorM_(ox). Finally, oxidized mediator M_(ox) is produced and electrons aretransferred to the electrode by the oxidation reduction reaction betweenreduced mediator M_(red) and the electrode. In other words, theelectrons generated by the enzyme reaction are rapidly transferred inhigh-volume from the enzyme to the electrode through the mediator. Atthis time, oxidation and reduction is repeatedly occurring between theenzyme E and the mediator M. The aforementioned enzyme reaction was forthe case where the substrate S is oxidized and electrons are transferredto the electrode, but if the substrate S is reduced and electrons areconsumed, the electrons will transfer to the enzyme from the electrodeby the reverse cycle.

FIG. 8 shows the structure of a mediator type biosensor with anelectrode which uses C₆₀ fullerene as shown in the Journal ofElectroanalytical Chemistry 454, 9-13, 1998. The C₆₀ fullerene 2, whichis the mediator, is fixed to the electrode 1 by a self organizingmonomolecular film 3 made from -HS—(CH₂)₂—NH₂. The surface of the C₆₀fullerene 2 is modified with ═CH—COOH in order for the C₆₀ fullerene 2and the self organizing monomolecular film 3 to bond. Furthermore, theenzyme 4 and the C₆₀ fullerene are not bonded, and the enzyme 4 issuspended in the solution.

With the biosensor described in the aforementioned documentation,electrons are generated by the oxidation reduction reaction between theglucose, which is the substrate 5, and the GOD, which is the enzyme 4,and the electrons are transferred to the electrode 1 through the C₆₀fullerene 2, and the glucose concentration is measured.

The mediator type biosensor shown in the aforementioned documentationuses C₆₀ fullerene 2, which has excellent properties for electronattracting and electron donating. However, the enzyme 4 is suspended inthe solution and is not bonded with the C₆₀ fullerene 2. Therefore, evenwhen electrons are transferred by the oxidation reduction reactionbetween the enzyme 4 and the substrate 5, if the enzyme 4 and the C₆₀fullerene 2 are not bonded, the C₆₀ fullerene 2 cannot transfer a largevolume of electrons at high-speed from the enzyme 4 to the electrode 1.

Furthermore, modifying groups are required on the surface of the C₆₀fullerene 2 in order to fix the C₆₀ fullerene 2 to the electrode 1.Therefore, the electron distribution of the Π bonds, which contribute tothe transmission of electricity, will be inconsistent, and there will beproblems with a loss of the properties of C₆₀ fullerene 2, includingelectron attracting and electron donating.

Therefore, an object of the present invention is to provide a targetrecognizing element wherein a receptor is fixed to an inclusion complexwhich includes a mediator.

Furthermore, another object of the present invention is to provide abiosensor wherein the position of the mediator is fixed with regard tothe electrode without using modifying groups on the surface of themediator.

DISCLOSURE OF THE INVENTION

In order to resolve the aforementioned problems, the first invention ofthe present application provides a target recognizing element comprisinga first host molecule having a hydrophilic group and an inclusion site,a second host molecule having hydrophilic groups and an inclusion site,a receptor which is bonded to the hydrophilic groups of the second hostmolecule and which reacts with the target, and a guest molecule which isincluded in the inclusion site of the first host molecule and in theinclusion site of the second host molecule, and which transmits a chargeproduced by the reaction between the target and the receptor.

The second host molecule and receptor are bonded together and thereceptor is fixed, so the reaction between the receptor and target canalways be performed close to the guest molecule which is included by thefirst host molecule and the second host molecule. Therefore, theelectric charge generated by this reaction can be transferred in largevolume and at high-speed by the guest molecule. Furthermore, the firstand second host molecules have hydrophilic groups, so even if the guestmolecule is insoluble, the target recognizing element can be used insolution, and for instance, the target recognizing element can easily bearranged on a substrate.

The second invention of the present application provides the targetrecognizing element according to the first invention, wherein the firsthost molecule is a first calixarene, the second host molecule is asecond calixarene, and the guest molecule is fullerene.

The inclusion sites of the first and second calixarenes include thefullerene by hydrophobic interaction and by Π-Π interaction, so theinsoluble fullerene which is the mediator can be made water-solublewithout using special modifying groups. Therefore, the distribution of Πelectrons which contribute to electric transmission will remainconsistent, and therefore the properties of fullerene, which has lowionization energy and high electron affinity, will not be lost.Therefore, the electric charge can be transferred in high-volume athigh-speed by the fullerene.

The third invention of the present application provides the targetrecognizing element according to the first invention, wherein thereceptor is one or a combination of a plurality of substances selectedfrom a group including enzymes, antibodies, DNA (deoxyribonucleic acid),and peptides.

Biological materials can be specifically captured using enzymes,antibodies, DNA, or peptides.

The fourth invention of the present application provides the targetrecognizing element according to the first or second invention, furthercomprising at least one layer of a polymer film between the second hostmolecule and the receptor.

The polymer film flattens the bonding interface between the second hostmolecule and the receptor without damaging the three-dimensionalstructure of the receptor, so loss of receptor activity can beprevented. Furthermore, the bonding area can be increased, so the bondbetween the second host molecule and the receptor can be made stronger.

The fifth invention of the present application provides the targetrecognizing element according to be fourth invention, wherein thepolymer film comprises a poly(diallyl dimethyl ammonium chloride) layerand a polyvinyl potassium sulfate layer.

The sixth invention of the present application provides the targetrecognizing element according to the first invention, and furthercomprises a polyion complex film which covers the receptor.

The bond between the receptor and the second host molecule can bestrengthened by the polyion complex film, so the durability of thetarget recognizing element can be increased.

The seventh invention of the present application provides the targetrecognizing element according to the first invention, wherein thereceptor is bonded to the hydrophilic groups of the second host moleculeunder conditions where the pH is between 4 and 8, and the temperature isbetween 15 and 45° C.

Loss of receptor activity can be prevented by bonding the receptor andthe second host molecule under the aforementioned conditions.

The eighth invention of the present application provides a biosensorcomprising a first host molecule having hydrophilic groups and aninclusion site, a second host molecule having hydrophilic groups and aninclusion site, an electrode to which the hydrophilic groups of thefirst host molecule are bonded, a receptor which is bonded to thehydrophilic groups of the second host molecule and which reacts with thetarget, and a guest molecule which is included in the inclusion site ofthe first host molecule and in the inclusion site of the second hostmolecule, and which transmits a charge produced by the reaction betweenthe target and the receptor to an electrode.

The receptor is fixed to the second host molecule, so the reactionbetween the receptor and the target can always be performed close to theguest molecule. Therefore, the electric charge generated by thisreaction can be transferred in high-volume and at high-speed from thereceptor to the electrode by the guest molecule. Furthermore, the guestmolecule is included by the first and second host molecules and issecurely fixed to the electrode, so modifying groups for fixing theguest molecule to the electrode are not required on the guest molecule.Therefore, the property of the guest molecule for transferring a highquantity of electrical charge at high-speed will not be lost.Furthermore, the first and second host molecules have hydrophilicgroups, so even if the guest molecule is insoluble, the biosensor caneasily be used in a solution.

The ninth invention of the present application provides the biosensoraccording to the eighth invention, further comprising detecting meansconnected to the electrode.

The charge which is transferred to the electrode can be measured by thedetecting means.

The 10th invention of the present application provides the biosensor ofthe eighth invention, wherein the first host molecule is the firstcalixarene, the second host molecule is the second calixarene, and theguest molecule is fullerene. The effect is similar to that of the secondinvention.

The 11th invention of the present application provides the biosensor ofthe eighth invention, wherein the receptor is one or a combination of aplurality of substances selected from a group including enzymes,antibodies, DNA, and peptides. The effect is similar to that of thethird invention.

The 12th invention of the present application provides the biosensoraccording to any one of the eighth through 10th inventions, furthercomprising at least one layer of a polymer film between the second hostmolecule and the receptor. The effect is similar to that of the fourthinvention.

The 13th invention of the present application provides the biosensor ofthe 12th invention, wherein the polymer film is comprising apoly(diallyl dimethyl ammonium chloride) layer and a polyvinyl potassiumsulfate layer. The effect is similar to that of the fifth invention.

The 14th invention of the present application provides the biosensor ofthe eighth invention, further comprising a polyion complex film whichcovers the receptor. The effect is similar to that of the sixthinvention.

The 15th invention of the present application provides the biosensor ofthe eighth invention, wherein the receptor is bonded to the hydrophilicgroups of the second host molecule under conditions in which the pH isbetween 4 and 8, and the temperature is between 15 and 45° C. The effectis similar to that of the seventh invention.

The 16th invention of the present application provides a detectionmethod for detecting the electrical charge produced from the reactionbetween the target and the receptor using the biosensor according toclaim 9.

Using the biosensor of the ninth invention, the electrical charge can beeffectively detected.

The 17th invention of the present application provides the detectionmethod according to the 16th invention, wherein the guest molecule isfullerene, and an excitation step to photoexcite the fullerene is alsoincluded.

The fullerene is photoexcited by radiating the fullerene with light, sothe high electron affinity, low ionization energy properties of thefullerene can be increased. Therefore, the reaction speed and thereaction sensitivity of the biosensor can be increased.

The 18th invention of the present application is a manufacturing methodfor the biosensor according to claim 8, comprising an inclusion complexproducing step of combining and mixing a solution containing the firsthost molecule and the second host molecule with a solution containingthe guest molecule to produce an inclusion complex comprising the firsthost molecule, the second host molecule, and the guest molecule; anelectrode forming step of bonding an anionic or a cationic molecule tothe surface of an electrode; an inclusion complex bonding step ofbonding the electrode formed in the electrode forming step with thehydrophilic groups of the first host molecule in the inclusion complex;and a receptor bonding step of bonding the receptor and the hydrophilicgroups on the second host molecule in the inclusion complex; wherein theguest molecule is included by the inclusion site of the first hostmolecule and the inclusion site of the second host molecule in theinclusion complex produced in the inclusion complex producing step. Abiosensor can be produced which has the same effect as the eighthinvention.

The 19th invention of the present application provides the biosensormanufacturing method of the 18th invention, wherein the hydrophilicgroup of the second host molecule and the receptor are bonded togetherunder conditions where the pH is between 4 and 8, and the temperature isbetween 15 and 45° C. in the receptor bonding step. The effect issimilar to that of the seventh invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a) is a diagram showing the basic structure of an electrode forthe mediator type biosensor of the present invention, and FIG. 1(b) isan explanatory diagram which describes the operation of the biosensor of(a).

FIG. 2 shows the structure of the biosensor according to a firstembodiment.

FIG. 3(a) is an example (1) of a biosensor with a detection unit. (b) isan example (2) of a biosensor with a detection unit.

FIG. 4(a) shows a structure (1) of the biosensor according to a thirdembodiment. (b) shows a structure (2) of the biosensor according to thethird embodiment.

FIG. 5 shows the structure of the biosensor according to the fourthembodiment.

FIG. 6 shows the electrical characteristics of the biosensor accordingto the first embodiment.

FIG. 7 is an explanatory diagram which describes the operatingprinciples of a mediator type biosensor.

FIG. 8 shows the structure of the electrode unit of a conventionalmediator type biosensor which uses C₆₀ fullerene.

BEST MODE FOR CARRYING OUT THE INVENTION

Basic Structure

FIG. 1(a) shows the basic structure of the electrode of the mediatortype biosensor of the present invention. The mediator type biosensor hasan electrode 11 and a target recognizing element 10 which is fixed onthe electrode 11. The target recognizing element 10 has an inclusioncomplex 13 and a receptor 15 fixed on the inclusion complex 13. Theinclusion complex 13 has a first host molecule 13 a, a second hostmolecule 13 b, and a guest molecule 13 c. The first guest molecule 13 ahas hydrophilic groups 19 a and an inclusion site 21 a, and similarly,the second host molecule 13 b also has hydrophilic groups 19 b and aninclusion site 21 b. The inclusion complex 13 has a structure such thatthe guest molecule 13 c is included by the inclusion site 21 a of thefirst guest molecule 13 a and the inclusion site 21 b of the second hostmolecule 13 b, and in whole is surrounded by the hydrophilic groups 19 aand 19 b. The included guest molecule 13 c functions as a mediator whichtransfers an electric charge from the receptors 15 to the electrode 11.For instance, fullerene may be suggested for the guest molecule 13 c andcalixarene as the first and second host molecules 13 a, 13 b.Furthermore, the first and second host molecules 13 a, 13 b may also bedifferent substances. Note, if the first and second host molecules 13 a,13 b are the same substance, creating the inclusion complex 13 will besimplified, which is preferable. For instance, if two types ofsubstances A, B are used as the first and second host molecules 13 a, 13b to produce the inclusion complex 13, three types of complexes will begenerated during preparation (AA, AB, BB). Therefore, these complexeswill need to be separated, and producing the inclusion complex 13 willbecome difficult. On the other hand, if the same substance is used,separation will not be necessary so production will be simplified.Furthermore, if the substances are the same, identifying the substanceof the first host molecule 13 a on the electrode 11 side and thesubstance of the second host molecule 13 b on the side opposite to theelectrode 11 will not be necessary, and the control of fixing theinclusion complex will be simplified.

The electrode 11 is ionic bonded by the electrostatic interaction of thehydrophilic groups 19 a of the first host molecule 13 a. The bondbetween the electrode 11 and the inclusion complex 13 is not restrictedto ionic bonds, and various other bonds such as covalent bonds andcoordinate bonds can be conceived. The electrode 11 may be any inertelectrode, and electrodes of Au, Ag, Pt, ITO, and carbon or the like maybe used. The use of carbon is preferable because carbon is inexpensive,can easily be formed, and provides a relatively stable electrode.

The receptor 15 is fixed to the inclusion complex 13 by the hydrophilicgroups 19 b of the second host molecule 13 b. The bond between thereceptor 15 and the hydrophilic groups 19 b is formed by ionic bonds orcovalent bonds, or the like. If the receptor 15 is one of an enzyme,antibody, DNA, cell, or peptide, or a combination thereof, biologicalsubstances can specifically be captured, and is therefore preferable.

FIG. 1(b) is a schematic diagram which describes the function of thebiosensor of FIG. 1(a). The target 23 is a substance which is capturedby the receptor 15. This biosensor functions as shown below. The target23 is captured by the receptor 15, and an oxidation reduction reactionoccurs between the target 23 and the receptor 15. Electrons e⁻ aregenerated by this oxidation reduction reaction, and for instance, thegenerated electrons e⁻ are transferred to the electrode 11 by the guestmolecule which is included by the first and second host molecules 13 a,13 b. The presence and quantity or the like of the target 23 can bemeasured by measuring the change in the electrical charge of theelectrode 11. In FIG. 1(b), the case where a negative charge istransferred is described, but a positive charge may also be transferred.

With this type of biosensor, the receptor 15 is fixed to the inclusioncomplex 13 by the bond between the second host molecule 13 b and thereceptor 15. Therefore, the reaction between the receptor 15 and thetarget 23 can always be performed in close proximity to the guestmolecule 13 c which is included by the first host molecule 13 a and thesecond host molecule 13 b. Therefore, the electrical charge produced bythis reaction can be transferred in high quantities and at high-speedfrom the receptor 15 to the electrode 11 by the guest molecule 13 c.Furthermore, the first and second host molecules 13 a, 13 b havehydrophilic groups 19 a, 19 b, so even if the guest molecule 13 c isinsoluble, the guest molecule 13 c can be used in the solution where thetarget 23 is measured. Furthermore, the target recognizing element 10can easily be arranged on a board or the like.

FIRST EMBODIMENT

FIG. 2 shows the structure of a biosensor according to a firstembodiment. The biosensor of the first embodiment will be describedbelow while referring to FIG. 1 and FIG. 2.

Structure of Biosensor

With the biosensor of the first embodiment, the target recognizingelement 10 wherein a receptor 15 is fixed to an inclusion complex 13 isfixed to the electrode 11. In this case, the guest molecule 13 c is C₆₀fullerene, and the first and second host molecules 13 a, 13 b includecalix[3]arene. Calix[3]arene is a structure with three phenolderivatives connected in a ring at the meta position, the oxygen atomsside of the phenol component is structured to be hydrophilic, and theC₆₀ fullerene is included at the inclusion site on the benzene ring sidewhich is opposite to the phenol component. Therefore, the inclusion siteis structured to be hydrophobic. The hydrophilic groups of thecalix[3]arene shown in FIG. 2 are modified using cationic quaternaryamines and have a positive electrical charge. Furthermore, the electrode11 is a metal and is modified by anionic carboxylic acid, and has anegative charge. Therefore, the electrode 11 and the hydrophilic groupsof the calix[3]arene are bonded by electrostatic interaction. Thereceptor 15 for instance is a lactate dehydrogenase, which is an enzymewhich reacts with pyruvic acid in the blood as the target. The lactatedehydrogenase, which has an acidic isoelectric point, has a negativeelectrical charge in a neutral aqueous solution, and is bonded to thehydrophilic groups of the calix[3]arene by electrostatic interaction.

In the above case, the charge on the hydrophilic groups of thecalix[3]arene was positive, and the charge on the surface of the enzymeand the surface of the electrode 11 was negative, but it is alsoacceptable for the charge on the hydrophilic groups of the calix[3]areneto be negative and the charge on the surface of the enzyme or thesurface of the electrode 11 to be positive. Furthermore, the bondbetween the receptor 15 and the inclusion complex 13 and the bondbetween the inclusion complex 13 and the electrode 11 may be a bondother than electrostatic interaction, such as a covalent bond.

The guest molecule 13 c, which is the mediator, may be a substance otherthan C₆₀ fullerene, and for instance higher-order fullerenes such asC₇₀, C₇₆, C₇₈, C₈₂, C₈₄, C₈₆, C₈₈, C₉₀, C₉₂, C₉₄, or C₉₆ are acceptable.Furthermore, if the fullerene includes a La atom, electrons will beprovided from the included element to the fullerene and the fullerenewill have surplus electrons, so not only will the fullerene haveincreased electrical conductivity, but the initial response time will beshorter, which is preferable.

Furthermore, in FIG. 2, the first and second host molecules 13 a, 13 bare both identical calix[3]arene, but it is also acceptable to usedifferent calixarene such as using calix[4]arene for the first hostmolecule 13 a and calix[3]arene for the second host molecule 13 b. Note,if the same calixarene is used, the inclusion complex 13 will be easierto produce as described above, and therefore this is preferable.

In the preceding example, the enzyme lactate dehydrogenase was used asthe receptor 15 in order to detect pyruvic acid, but other enzymes maybe used depending on the target to be detected. Other enzymes which maybe used include for instance, oxidases (such as glucose oxidase),dehydrogenases (such as alcohol dehydrogenase), reductases (such asadrenodoxin), oxygenases, hydroperoxygenases (such as catalase), urease,creatinine deaminase, or the like. If urease is used as the enzyme,blood urea nitrogen (BUN) can be measured, and if creatinine deaminaseis used, then creatinine can be measured, and thus kidney disease can bediagnosed.

With the aforementioned biosensor, the C₆₀ fullerene is included by theΠ-Π interaction and the hydrophobic interaction of the inclusion sitesof the calix[3]arene, which is the first and second host molecule 13 a,13 b, and thus stably fixed to the electrode 11. At this time, the C₆₀fullerene is included by the calix[3]arene in the suspended state, andmodifiers are not bonded. Therefore, the distribution of Π electronswhich contribute to electrical conductivity will remain consistent, sothe high electron affinity and low ionization energy properties offullerene will not be lost. Therefore, a large electric charge canrapidly be transferred from the receptor to the C₆₀ fullerene and theelectrode. Furthermore, the side opposite to the inclusion site of thecalix[3]arene is constructed with hydrophilic phenol, so water insolubleC₆₀ fullerene can be made soluble without using modifying groups by theinclusion of the C₆₀ fullerene in the calix[3]arene. Therefore, thebiosensor can easily be used in a solution.

Detection Method Using Biosensor

FIG. 3(a), (b) show an example of the biosensor with a detecting unit.In FIG. 3(a), the target recognizing element 42 from FIG. 2 is fixed toan electrode 40, and the electrode 40 is connected to a detecting unit45. A test sample is dripped onto the region of the electrode 40 wherethe target recognizing element 42 is fixed, the current is measured bythe detecting unit 45, and the concentration or the like of the targetin the test sample is calculated.

On the other hand, in FIG. 3(b), the tip of the electrode 40 to whichthe target recognizing element 42 has been fixed is immersed in the testsolution, and the current is measured by the detecting unit 45.

Note, if external light is radiated on the target recognizing element 42while measuring, the C₆₀ fullerene will be photoexcited, so the highelectron affinity and low ionization energy properties of the fullerenecan be increased. Therefore, the response speed of the targetrecognizing element will be faster, and the reaction sensitivity can beincreased. The wavelength band where fullerene becomes photoexcited isbroad, but in particular, excitation is more efficient at wavelengths ator below 620 nm. Red LED and Ar laser may be used as light sources whichhave this wavelength.

Biosensor Manufacturing Method

The biosensor of the first embodiment is manufactured as shown below.Calix[3]arene and C₆₀ fullerene are mixed in an aqueous solution and themixture is agitated. Ultrasonic treatment is preferably used for theagitation. Because of this treatment, the C₆₀ fullerene will be includedby the hydrophobic groups which are the inclusion site of thecalix[3]arene, to produce an inclusion complex 13. The electrode 11 andthe hydrophilic groups of the calix[3]arene are modified with anionic orcationic molecules. At this time, the bond between the electrode 11 andthe calix[3]arene and the bond between the calix[3]arene and the lactatedehydrogenase are modified to bond by electrostatic interaction. Themodified inclusion complex 13, electrode 11, and the lactatedehydrogenase are bonded together to obtain a biosensor which is capableof measuring pyruvate acid which is the target. At this time, if thebond between the lactate dehydrogenase and the inclusion complex 13 isformed under conditions where the pH is between 4 and 8 and thetemperature is between 15 and 45° C., loss of enzymatic activity can beprevented, and therefore this is preferable.

EXPERIMENTAL EXAMPLE 1

A experimental example in which an inclusion complex was created byincluding C₆₀ fullerene in calix[3]arene, and lactate dehydrogenase wasfixed to the inclusion complex as the receptor, is shown below.

(1) Synthesis of Calix[3]arene

First, using the triester derivatives of calix[3]arene as a rawmaterial, an excess of N, N-dimethylpropane diamine was added tosynthesize the precursor of the calix[3]arene using aminolysis. Thisprecursor was N-methylated using dimethyl sulfate to synthesizecalix[3]arene with quaternary amines on the end.

(2) Preparation of Calix[3]arene and C₆₀ Fullerene

C₆₀ fullerene (72 mg, 0.1 mmol) was mixed into an aqueous solution ofcalix[3]arene (10 ml, 0.5 mmol/dm³) synthesized as shown above, andafter repeated agitation and ultrasonic treatment, the undissolvedfullerene was removed by centrifugal separation, to prepare an aqueoussolution of the inclusion complex which was the calix[3]arene and C₆₀fullerene complex. The width of the inclusion complex produced wasapproximately 1 nm, and the height was approximately 2 nm.

(3) Preparation of the Electrode

On the other hand, an electrode 11 with anionic molecules on the surfacewas created by immersing the metal electrode in an ethanol solutioncontaining sodium 2-mercaptoethane sulfonate.

(4) Fixing the Inclusion Complex

The inclusion complex is tightly arranged on the electrode by immersingthe electrode prepared according to (3) above in an aqueous solution ofinclusion complex (0.25 mmol/dm³) in order to produce a biosensor. Atthis time, the inclusion complex was bonded to the electrode byelectrostatic interaction.

(5) Fixing the Receptor

Next, the electrode with the inclusion complex was immersed for 20minutes at room temperature in a HEPES(2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) bufferedsolution (pH=7.0) containing lactate dehydrogenase (0.2 mg/ml), and theenzyme was fixed to the inclusion complex by electrostatic interaction.

The electrical characteristics obtained using the cyclic voltammetrymeasurement method of the biosensor of the first embodiment obtained bythe aforementioned manufacturing method is shown in FIG. 6. This is theproperty diagram when a biosensor chip to which lactate dehydrogenase(LDH) was fixed is immersed in a solution of 125 μM of reducednicotinamide adenine dinucleotide (NADH) and between 0 and 250 μM ofpyruvic acid. A biosensor chip which is sensitive enough to measureminute quantities of pyruvic acid could be obtained.

SECOND EMBODIMENT

A biosensor with the structure shown in the first embodiment wherein thereceptor 15 is an antibody, DNA, cell, or peptide will be described.

Antibody

If the receptor 15 is an antibody, the target will be antigens whichspecifically react with the receptor 15, and the existence andconcentration or the like of the target can be measured. The types ofantigen include viruses, bacteria, pollen, mold, and dust mites or thelike. To illustrate, a case where the antibody is “Mouse IgG” and theantigen is “Protein A” will be described.

First, the antigen “Protein A” is injected into a mouse, and “Mouse IgG”antibody is produced using the immuno response of the mouse. Theantibody “Mouse IgG” is extracted, purified, and fixed to the inclusioncomplex 13. The Fc region of the mouse IgG is modified with a carboxylgroup or an amino group or the like in order to fix to the inclusioncomplex by electrostatic interaction or covalent bonding. When Protein Ais injected therein, the Protein A and the Mouse IgG will specificallybond. The electric charge produced from the Protein A made from polarmolecules will reach the electrode 11 through the antibody and theinclusion complex 13. The quantity of the antigen Protein A can bedetermined by measuring the current flowing to the electrode 11.

DNA

A method to detect target DNA, for a case where the receptor 15 is probeDNA which specifically reacts with the DNA that is the target, will bedescribed.

DNA has a double helical structure, but when used as the receptor 15 forthe biosensor, a single strand is used. First, a single strand DNA whichhas the base sequence and complementation as the target DNA isartificially organically synthesized to produce the probe DNA. Thisprobe DNA is fixed on the inclusion complex 13. Next, the desired DNA isextracted and purified from a biological sample, and heated to 95° forinstance to obtain a single strand. If this DNA and the base sequence ofthe probe DNA are complementary, or in other words if the DNA in thetest sample has the base sequence of the DNA to be detected, both DNAwill combine and form a double helix. The double helix structure will bespecifically bonded, and an intercalator which is the source of theelectrical charge is inserted in the gap in the double helicalstructure. Therefore, the presence of a double helical structure, or inother words, whether or not the DNA in the test sample has the targetbase sequence, can be determined by changes in the current flowing tothe electrode 11.

Cells

The method for detecting an antigen, which is the target, when thereceptor 15 is a cell will be described. Immunocyte such as NK cells orB cells are extracted and purified from an organism, and then fixed tothe inclusion complex 13. If a protein molecule terminal group which isbonded to the cell surface is used to fix the cell to the inclusioncomplex 13, the fixing can be easily performed, and this is preferable.The antigen may be a virus, bacteria, pollen, mold, or dust mite or thelike. When the biosensor to which the cell is fixed is introduced to thetest sample, the cell which is the receptor 15 will attack the antigenand the antigen will be disintegrated by the enzymes in the cell. Theelectrical charge produced by the disintegration process will betransferred to the electrode 11 through the inclusion complex 13. Thequantity of antigen can be determined by measuring the electricalcurrent flowing to the electrode 11 at this time.

Peptides

The method for detecting a peptide, which is the target, when thereceptor 15 is a peptide will be described. A probe peptide whichspecifically reacts with the desired target is created in a phage usinggenetic engineering methods. This peptide is extracted, purified, andfixed to the inclusion complex 13. When a biosensor which contains thisinclusion complex 13 is injected into the test sample, the probe peptideand a peptide which corresponds to the charge of the side groups of theamino acids which make up the probe peptide, will bond together. At thistime, the target peptide can be detected and quantified by measuring theelectrical current flowing to the electrode 11.

THIRD EMBODIMENT

FIGS. 4(a), (b) show the structure of the biosensor of the thirdembodiment. Flag numbers which are identical to those in FIG. 1 identifysimilar structural elements as those in the first embodiment. Thebiosensor of FIG. 4(a) comprises the target recognizing element 10 ofFIG. 1 with a polymer film 50. The polymer film 50 is establishedbetween the second host molecule 13 b and the receptor 15. This polymerfilm 50 flattens the contact interface between the second host molecule13 b and the receptor 15 without damaging the three-dimensionalstructure of the receptor 15, so a loss of activity of the receptor 15can be prevented. Furthermore, the contact surface area can beincreased, so the second host molecule 13 b and the receptor 15 can bemore tightly connected.

As shown in FIG. 4(b), the polymer film 50 may be a polymer film 52, 54with a double layer structure. For example, PDDA (poly(diallyl dimethylammonium chloride)) 52 which is a cationic polymer film is formed on theinclusion complex 13, and PVS (polyvinyl potassium sulfate) 54 which isan anionic polymer film is formed thereon, thus fixing the enzyme to theinclusion complex 13. In this manner, the polymer film 50 will have ananionic and cationic double layer structure, so a positively chargedenzyme can be fixed in place of the negatively charged enzyme.Furthermore, the polymer film 50 may also have two or more layers.

EXPERIMENTAL EXAMPLE 2

An experimental example in which an inclusion complex was created byincluding C₆₀ fullerene in calix[3]arene, and then a polymer film andlactate dehydrogenase as the receptor were fixed to the inclusioncomplex, is shown below.

(1) Synthesis of Calix[3]arene

First, using the triester derivatives of calix[3]arene as a rawmaterial, an excess of N, N-dimethylpropane diamine was added tosynthesize the precursor of the calix[3]arene using aminolysis. Thisprecursor was N-methylated using dimethyl sulfate to synthesizecalix[3]arene with quaternary amines on the end.

(2) Preparation of Calix[3]arene and C₆₀ Fullerene

C₆₀ fullerene (72 mg, 0.1 mmol) was mixed into an aqueous solution ofcalix[3]arene (10 ml, 0.5 mmol/dm³) synthesized as shown above, andafter repeated agitation and ultrasonic treatment, the undissolvedfullerene was removed by centrifugal separation, to prepare an aqueoussolution of the inclusion complex which was the calix[3]arene and C₆₀fullerene complex. The width of the inclusion complex produced wasapproximately 1 nm, and the height was approximately 2 nm.

(3) Preparation of the Electrode

On the other hand, an electrode 11 with anionic molecules on the surfacewas created by immersing a metal electrode in an ethanol solutioncontaining sodium 2-mercaptoethane sulfonate.

(4) Fixing the Inclusion Complex

The inclusion complex is tightly arranged on the electrode by immersingthe electrode prepared according to (3) above in an aqueous solution ofinclusion complex (0.25 mmol/dm³) in order to produce a biosensor. Atthis time, the inclusion complex was joined to the electrode byelectrostatic interaction.

(5) Fixing the Polymer Film

The inclusion complex which was fixed on the electrode was immersed for20 minutes at room temperature in a HEPES buffered solution (pH=7.0)containing PDDA (6 mg/ml), and a PDDA film was formed on the inclusioncomplex by electrostatic interaction. After washing in purified water,the electrode was immersed for 20 minutes at room temperature in a HEPESbuffered solution (pH=7.0) containing PVS (4 mg/ml) to form a PVS layeron the PDDA layer by electrostatic interaction.

(6) Fixing the Receptor

Next, the electrode with the inclusion complex was immersed for 20minutes at room temperature in a HEPES(2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) bufferedsolution (pH=7.0) containing lactate dehydrogenase (0.2 mg/ml), and theenzyme was fixed on the inclusion complex by electrostatic interaction.

FOURTH EMBODIMENT

FIG. 5 shows the structure of the biosensor of the fourth embodiment.Flag numbers which are identical to those in FIG. 1 identify similarstructural elements as those in the first embodiment. The biosensor ofFIG. 5 comprises the target recognizing element 10 of FIG. 1 with apolyion complex film 56. The polyion complex film 56 is formed over thereceptor 15, and strengthens the bond between the receptor 15 and thesecond host molecule 13 b. Therefore, the durability of the biosensorcan be increased. The polyion complex film 56 may be for instance apolyion complex film such as cationic poly-L-lysine or anionic glutamateor acrylate.

EXPERIMENTAL EXAMPLE 3

An experimental example in which an inclusion complex was created byincluding C₆₀ fullerene in calix[3]arene, and then lactate dehydrogenaseas the receptor was fixed to the inclusion complex, is shown below.

(1) Synthesis of Calix[3]arene

First, using the triester derivatives of calix[3]arene as a rawmaterial, an excess of N, N-dimethylpropane diamine was added tosynthesize the precursor of the calix[3]arene using aminolysis. Thisprecursor was N-methylated using dimethyl sulfate to synthesizecalix[3]arene with quaternary amines on the end.

(2) Preparation of Calix[3]arene and C₆₀ Fullerene

C₆₀ fullerene (72 mg, 0.1 mmol) was mixed into an aqueous solution ofcalix[3]arene (10 ml, 0.5 mmol/dm³) synthesized as shown above, andafter repeated agitation and ultrasonic treatment, the undissolvedfullerene was removed by centrifugal separation, to prepare an aqueoussolution of the inclusion complex which was the calix[3]arene and C₆₀fullerene complex. The width of the inclusion complex produced wasapproximately 1 nm, and the height was approximately 2 nm.

(3) Preparation of the Electrode

On the other hand, an electrode 11 with anionic molecules on the surfacewas created by immersing a metal electrode in an ethanol solutioncontaining sodium 2-mercaptoethane sulfonate.

(4) Fixing the Inclusion Complex

The inclusion complex is tightly arranged on the electrode by immersingthe electrode prepared according to (3) above in an aqueous solution ofinclusion complex (0.25 mmol/dm³) in order to produce a biosensor. Atthis time, the inclusion complex was joined to the electrode byelectrostatic interaction.

(5) Fixing the Receptor

Next, the electrode with the inclusion complex was immersed for 20minutes at room temperature in a HEPES(2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) bufferedsolution (pH=7.0) containing lactate dehydrogenase (0.2 mg/ml), and theenzyme was fixed on the inclusion complex by electrostatic interaction.

(6) Fixing the Polyion Complex Film

After fixing the enzyme to the inclusion complex, a polyion complex filmis formed on the surface of the enzyme using a method of casting a 1 mMsolution of poly-L-lysine.

INDUSTRIAL APPLICABILITY

Using the present invention, a target recognizing element can beprovided wherein a receptor is fixed to an inclusion complex containinga mediator.

1. A target recognizing element comprising: a first host molecule havinga hydrophilic group and an inclusion site; a second host molecule havinga hydrophilic group and an inclusion site; a receptor which is bonded tothe hydrophilic group of the second host molecule and which reacts withthe target; and a guest molecule which is included in the inclusion siteof the first host molecule and in the inclusion site of the second hostmolecule, and which transmits a charge produced by the reaction betweenthe target and the receptor.
 2. The target recognizing element accordingto claim 1, wherein the first host molecule is a first calixarene, thesecond host molecule is a second calixarene, and the guest molecule isfullerene.
 3. The target recognizing element according to claim 1,wherein the receptor is one or a combination of a plurality ofsubstances selected from a group including enzymes, antibodies, DNA(deoxyribonucleic acid), and peptides.
 4. The target recognizing elementaccording to claim 1 further comprising at least one layer of a polymerfilm between the second host molecule and the receptor.
 5. The targetrecognizing element according to claim 4, wherein the polymer film iscomprising a poly(diallyl dimethyl ammonium chloride) layer and apolyvinyl potassium sulfate layer.
 6. The target recognizing elementaccording to claim 1, further comprising a polyion complex film whichcovers the receptor.
 7. The target recognizing element according toclaim 1, wherein the receptor is bonded to the hydrophilic group of thesecond host molecule under conditions in which the pH is between 4 and8, and the temperature is between 15 and 45° C.
 8. A biosensor,comprising: a first host molecule having a hydrophilic group and aninclusion site; a second host molecule having a hydrophilic group and aninclusion site; an electrode to which the hydrophilic groups of thefirst host molecule are bonded; a receptor which is bonded to thehydrophilic group of the second host molecule and which reacts with thetarget; and a guest molecule which is included in the inclusion site ofthe first host molecule and in the inclusion site of the second hostmolecule, and which transmits a charge produced by the reaction betweenthe target and the receptor to an electrode.
 9. The biosensor accordingto claim 8, further comprising detecting means connected to theelectrode.
 10. The biosensor according to claim 8, wherein the firsthost molecule is the first calixarene, the second host molecule is thesecond calixarene, and the guest molecule is fullerene.
 11. Thebiosensor according to claim 8, wherein the receptor is one or acombination of a plurality of substances selected from a groupconsisting of enzymes, antibodies, DNA, and peptides.
 12. The biosensoraccording to claim 8, further comprising at least one layer of a polymerfilm between the second host molecule and the receptor.
 13. Thebiosensor according to claim 12, wherein the polymer film comprises apoly(diallyl dimethyl ammonium chloride) layer and a polyvinyl potassiumsulfate layer.
 14. The biosensor according to claim 8, furthercomprising a polyion complex film which covers the receptor.
 15. Thebiosensor according to claim 8, wherein the receptor is bonded to thehydrophilic group of the second host molecule under conditions in whichthe pH is between 4 and 8, and the temperature is between 15 and 45° C.16. A detection method, comprising the step of detecting the electricalcharge produced from the reaction between the target and the receptorusing the biosensor according to claim
 9. 17. The detection methodaccording to claim 16, wherein the guest molecule is fullerene, andfurther comprising the step of photoexciting the fullerene.
 18. A methodof manufacturing the biosensor according to claim 8, comprising thesteps of: combining and mixing a solution containing the first hostmolecule and the second host molecule with a solution containing theguest molecule to produce an inclusion complex comprising the first hostmolecule, the second host molecule, and the guest molecule; bonding ananionic or a cationic molecule to the surface of an electrode; bondingthe electrode formed in the electrode forming step with the hydrophilicgroup of the first host molecule in the inclusion complex; and bondingthe receptor and the hydrophilic group on the second host molecule inthe inclusion complex; wherein the guest molecule is included in theinclusion complex produced in the inclusion complex producing step bymeans of the inclusion site of the first host molecule and the inclusionsite of the second host molecule.
 19. The biosensor manufacturing methodaccording to claim 18, wherein the hydrophilic group of the second hostmolecule and the receptor are bonded together under conditions in whichthe pH is between 4 and 8, and the temperature is between 15 and 45° C.,in the receptor bonding step.